1. Field of the Invention
This invention relates to an effective vaccine against viral hepatitis which is characterized by containing large particles of filamentous and/or Dane particles characterized by a particle size of 20 to 50 nanometers (nm) said vaccine being further characterized by the substantial absence of antibodies to the antigens therein, the vaccine having both hepatitis B surface antigens as well as antigens which are specific for the virion itself such as e-antigen(s). The invention is also directed to the preparation of such vaccine, its purification and the multiplication of the vaccine in a host animal and the use thereof in active immunization against various forms of viral hepatitis.
DISSUSSION OF THE PRIOR ART
Epidemiologic and human volunteer studies carried out between 1939 and 1967 prove the existence of two major hepatitis viruses--virus A and virus B. It became increasingly clear that further understanding of the virology of these viruses and the prevention of the diseases require the development of immunologic methodology. Considerable research focused on these viruses, particularly that known as virus B. Virus B was associated with those patients who had undergone a number of injections or transfusion of blood and, hence, the virus became known as serum hepatitis. As a result of the discovery of antigens specific for hepatitis B virus infections [Prince et al., Am. J. Hyg. 79, 365, 1964; Blumberg et al., JAMA 191, 541, 1965; Prince, Proc. Nat. Acad. Sci. USA,
60, 814, 1968] it became apparent that "serum hepatitis virus" was, in fact, contagious under certain circumstances. It therefore became appropriate to alter the terminology. The Committee on Viral Hepatitis of the Division of Medical Sciences, Academy of Sciences, National Research Council therefore recommended in 1972 a return to the former terminology, namely hepatitis A virus for infectious hepatitis virus and a hepatitis B virus for serum hepatitis virus. At the same time the committee recommended the use of the term hepatitis B antigen (HB.sub.s Ag) for the antigen in place of the previous term, e.g., Australia antigen (Au Ag), which had been employed inasmuch as the antigen had first been discovered in the blood of Australian aboriginies; Serum Hepatitis Antigen (SH); Hepatitis Associated Antigen (HAA); etc.
Subsequently, studies focused upon the antigenic specificities of hepatitis B virus infections and research centered about sub-type antigens now termed HB.sub.s Ag/adywr, etc., the hepatitis B core antigen (HB.sub.c Ag) discovered by Almeida, the e-antigen(s) discovered by Magnius and Espmark as well as hepatitis B surface antigen (HB.sub.s Ag). Antibody to HB.sub.s Ag is termed Anti-HB.sub.s.
Many of the early researchers, including Blumberg, were of the belief that hepatitis B antigens were of host genetic origin. Other researchers, including Prince and Vnek, considered that hepatitis B antigens were determined by the genome of hepatitis B virus. It became increasingly evident that active immunization would be required to prevent infections by this virus, and to that end research focused upon the preparation of a vaccine.
Heretofore, particles in human sera having a particle size in the range of about 20 to 25 nm were regarded as the predominant particles which carry the hepatitis B surface antigens. Researchers focused about the separation of these particles from the other proteinaceous material in the blood, and especially from the larger 42 nm Dane particle which many suspected to be the infective virion. It was postulated that if the antigen could be purified to the required extent, and Dane particles removed, there could be provided a material which when diluted with a physiologically acceptable medium and further inactivated for safety, would provide a vaccine for active immunization. To this end Blumberg in U.S. Pat. No. 3,636,191 recommended the treatment of blood plasma from HB.sub.s Ag carriers with a mixture of enzymes to digest new particle-associated proteins, followed by conventional centrifugation steps to recover purified hepatitis B surface antigen on particles having a particle size of about 22 nm. Blumberg et al. had recognized that the 22 nm hepatitis B surface antigen-associated particles were in the form of a shell which had no core and which was substantially resistant to the various enzymes employed in the digestion procedure. Blumberg provided a vaccine having a density of the order of 1.21 Gm/cc which, according to Blumberg, can be diluted with a physiologically acceptable medium and employed as an active immunizing agent.
The vaccine provided by Blumberg may be effective in inducing synthesis of antibodies in a host animal such as a human being, however, such antibodies are now known by us to be of limited specificity and do not include antibodies to the unique specificity(s), e.g., e-antigen, present only on the larger particles, such as the Dane particle (i.e., the virion). The vaccine proposed by Blumberg is thus not protective against the large doses of virus to which people are frequently exposed, e.g. in transfusions. (See FIGS. 1 and 2 which show the theoretical basis for this conclusion). While it would theoretically be possible to produce very high titers of anti-HBs no acceptable means for doing this in man has been provided. Theoretically one could employ Freund's adjuvant to stimulate production of high quantities of antibodies. Then the very high concentrations of anti-HBs might effectively neutralize both hepatitis B surface antigen and the e-antigen-associated virions (see FIG. 2). However, because of potential carcinogencity, and other adverse side effects of Freund's and other known adjuvants, no method is presently known for the production of such high titers of anti-HBs in man.
It has therefore become desirable to provide a vaccine against viral hepatitis which not only stimulates and induces the production of anti-HBs, but also that of antibodies specific to the virion itself, i.e., the 42 nm Dane particle, such as e-antigen(s). Production of such a vaccine has previously been impossible when pools of human HB.sub.s Ag-containing plasma are used as source material. The reason for this is that such plasma pools contain an excess of anti-e antibody. This combines with any e-antigen-containing particles, resulting in their precipitation. The precipitated e-antigen-containing particles are difficult if not impossible to purify by conventional methods, and therefore do not appear in the final product.
It is therefore an object of this invention to provide a means for isolating hepatitis B surface antigen together with free uncombined and unprecipitated e-antigen-containing particles to provide a vaccine for use in active immunization. It is a further object of this invention to provide a means for the multiplication of such antigens in a host animal, and followed by purification of the HB-associated antigenic material from the host animals blood to produce a broad and effective vaccine against viral hepatitis and related disorders.